This is an application to dissect at the molecular level the virus regulatory mechanisms that are responsible for controlling the replication cycle of herpes simplex virus. We propose to approach this problem by constructing cell lines that contain and express individual members of the immediate early gene family to deterine which of these genes has a role in facilitating the transition from the synthesis of immediate early to early transcription. Cell lines containing these genes will be constructed by transformation of primate and murine cells with selectable markers and a cloned fragment of HSV DNA. Cells containing and expressing the cotransformed sequence will be identified on the basis of their ability to support the replication of ts mutants and to promote transcription of transfected virus DNA templates. The availability of cell lines expressing discrete virus functions will permit us to isolate mutants in these genes and to begin to unravel their function. A second aim of this application is to use eukaryotic expression vectors to attempt to produce large amounts of immediate early gene products. This will facilitate purification of these proteins so that we can begin to study their mechanism of action. In another study we propose to identify sequences that have been juxtaposed to promoterless tk genes to ask why are these genes transcribed when cells transformed with promoterless sequences are infected with HSV. Finally, we are constructing hybrid viruses that contain eukaryotic genes in the body of the HSV chromosome. These chimeric viruses are being used to introduce foreign DNA sequences into eukaryotic cells and for studies on the expression of eukaryotic sequences when they are cis to the virus chromosome.